PROJECT SUMMARY Theiler?s murine encephalomyelitis virus (TMEV) infection of susceptible mice leads to the establishment of a persistent virus infection the central nervous system (CNS). The persistent TMEV infection is associated with an inflammatory response that leads to the development of a chronic, progressive demyelinating disease. TMEV remains persistent in microglia cells which are the CNS resident immune cell population. We have previously shown that TMEV- infected microglia become activated to secrete pro-inflammatory cytokines, chemokines, and effector molecules. TMEV-induced demyelinating disease serves as a mouse model for multiple sclerosis (MS) in humans which is a demyelinating disease of the CNS associated with an inflammatory immune response. Activated microglia have been identified in both early and late demyelinating lesions in MS patients, and thus have been suggested to play a role in the inflammatory immune response. The causative agent for MS has not been determined, however epidemiological studies have suggested that a virus infection during childhood may begin the disease process where clinical signs of disease do not appear until adulthood. The proposed mechanism by which virus infections may trigger demyelinating disease is through bystander damage and bystander activation. Recently we have determined that TMEV- infected microglia secrete exosomes. Exosomes are microvesicles released by cells that are taken up by other cells and thus represent a means of cell to cell communication. Exosomes package mRNA, miRNA, DNA, and proteins from the cytoplasm of the cell which are then transferred to the cytoplasm of the recipient cell. We have recently determined that exosomes secreted by microglia during TMEV infection contain viral RNA, thus we propose that exosomes may contribute to persistent virus infection via transfer of viral RNA to bystander cells. We further propose that the viral RNA in exosomes secreted by microglia during TMEV infection activate bystander CNS cells to promote the inflammatory response associated with development and progression of demyelinating disease. We will first determine whether the viral RNA in exosomes secreted by virus infected microglia are transferred to uninfected cells and replicate to maintain virus infection. We will further determine whether the viral RNA in exosomes secreted by microglia during TMEV infection activate bystander CNS cells, such as microglia, astrocytes, and neurons, via innate immune receptors to express pro-inflammatory cytokines, chemokines, and effector molecules. Most importantly, we will determine whether viral RNA in exosomes secreted by microglia during persistent TMEV infection can maintain viral persistence and activate an inflammatory immune response that promotes development and progression of demyelinating disease in nave mice. These studies may provide a new mechanism by which viral RNA in exosomes secreted by microglia may be mediating disease pathogenesis and may provide a new target for development of therapies for neurological diseases.